PROJECT SUMMARY The ability to resurface larger defects (>2cm2 up to a hemicondyle) with mature hyaline articular cartilage and address the underlying bone deficit in a single procedure makes osteochondral allograft transplantation an attractive option. The appropriate size and surface contour can be matched when the graft is obtained from an appropriately selected organ donor. Mature chondrocytes can survive for many years post- transplantation without immunosuppressive therapy. As there is insufficient supply of suitable cartilage grafts to meet the clinical demand, the development of tissue engineered osteochondral grafts and strategies to promote their successful application in the joint would have significant clinical impact for treatment of localized cartilage lesions (of focus in this proposal) and whole joint surfaces (to be addressed in the future). Data from animal studies and early clinical trials suggest that early inhibition of the intra-articular inflammatory response (e.g., 4 weeks) posttraumatic injury of the knee may improve clinical outcomes. Research from our team portrays dexamethasone (dex), a synthetic glucocorticoid that has pro-anabolic and anti-catabolic effects in cartilage tissue engineering systems, as a critical element for cultivating cartilage tissues with native properties, as well as for providing chondroprotection to inflammatory cytokines. As these factors are likely to impact the clinical success of cartilage tissue engineering strategies, and dex is FDA- approved and used clinically to reduce pain and inflammation, we sought to develop a strategy to make these tissue culture findings more clinically relevant. From our perspective, an ideal method would retain the benefits of dex on engineered cartilage without the requirement for its exogenous supplementation, as clinical injections of steroids in the joint have been associated with negative side effects. In this new R01 grant, we propose the incorporation of dex-supplemented, poly(lactide-co-glycolide) (PLGA) microspheres into cell-seeded hydrogel constructs as a means for dex delivery from within engineered cartilage. We present preliminary data demonstrating that dex release internally from PLGA microspheres incorporated in chondrocyte-seeded hydrogel constructs promotes growth of mechanically functional cartilage tissue and confers cytokine protection in a manner akin to that observed with dex externally supplemented in culture media. In vivo efficacy of dex-microspheres has been confirmed by our team for adipose tissue engineering applications. With localized dex delivery, these benefits were achievable using concentrations that are orders of magnitude lower than adopted for clinical administration by injection. To build on this promising finding and to further realize the potential for clinical translation of this strategy for cell-based cartilage repair, we pose the following global hypothesis: Incorporation of polymer microspheres that release dex from within cell-seeded hydrogel constructs will protect constructs from the deleterious effects of cytokine exposure and improve cartilage repair in an inflammatory environment.